As a first line of defence in inflammatory conditions, cellular components of the innate immunity release pro-inflammatory cytokines. The endothelium, lining the vessels and constituting the barrier between blood and tissue, becomes activated by these inflammatory factors, resulting in for instance the upregulation of adhesion molecules, generation of reactive oxygen species (ROS) and increased vascular permeability. The activation of the endothelium results in the recruitment of inflammatory cells and increased vascular leakage. These responses contribute to tissue oedema and organ failure in systemic inflammatory conditions, such as sepsis or SIRS (systemic inflammatory response syndrome). In chronic inflammatory settings, this promotes endothelial dysfunction and subsequent cardiovascular disease. We have demonstrated the tyrosine phosphatase SHP-2 to be inactivated in endothelial cells under inflammatory conditions, resulting in increased expression of ICAM-1 and VCAM-1 and subsequently in enhanced endothelial leukocyte recruitment and transmigration in vitro and in vivo. Importantly, re-installing SHP-2 activity prevented these inflammatory changes. Moreover, we found inflammatory vascular permeability to be enhanced in mice treated with an SHP-2 inhibitor, pointing towards a protective role of SHP-2 in this context. These data demonstrate that an inactivation of SHP-2 may indeed drive endothelial dysfunction and promote vascular events. Ongoing projects aim to further characterize the underlying mechanisms and direct targets of SHP-2 within this context as well as investigating the function of SHP-2 in endothelial activation and dysfunction in cardiovascular disease under insulin resistant conditions mimicking type 2 diabetes.

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The formation of new blood vessels (angiogenesis) plays an essential role not only in physiological processes such as development (embryogenesis) and wound healing, but also in pathophysiological processes such as tumour growth and diabetic retinopathy. A condition, which is a strong inducer of angiogenesis is hypoxia. Hypoxia induces the accumulation and activation of the α subunit of the transcription factor hypoxia inducible factor 1 (HIF-1), which is otherwise rapidly degraded by the proteasome under normoxic conditions. HIF-1 induces expression of several angiogenic genes, thus being the target for therapeutic strategies. We found that downregulation of the tyrosine phosphatase SHP-1 enhanced HIF-1α accumulation and increased hypoxia induced VEGF expression as well as endothelial cell (EC) proliferation. Interestingly, we previously showed that the SHP-1 homologue SHP-2 is additionally important for angiogenesis. In contrast to SHP-1, down regulation or inhibition of SHP-2 impairs growth factor mediated EC proliferation, migration and vessel sprouting in vitro and ex vivo. In follow-up projects, we observed that overexpression of an inactive SHP-2 mutant in EC under hypoxia led to reduced HIF-1α accumulation and activity, demonstrating that SHP-2 activity is essential for HIF-1α activity. Moreover, we were able to modulate hypoxic wound healing angiogenesis in vivo by site-directed gene transfer using magnetic lentiviruses expressing different SHP-2 constructs. Whereas introduction of an inactive SHP-2 mutant impaired wound healing angiogenesis, expression of a constitutively active SHP-2 mutant accelerated this. We found that SHP-2 promotes hypoxic angiogenesis by preventing HIF-1α degradation via a Src dependent mechanism. In a further study, we detected that SHP-2 influences 26S proteasomal activity under hypoxia in vitro and in vivo. We are now interested in studying the mechanism of SHP-2 dependent regulation of proteasomal activity under hypoxia in more detail as well as a possible role for SHP-2 in hypoxia dependent vascular permeability and the identification of the underlying mechanisms. Additionally, ongoing projects investigate the role of SHP-2 for angiogenesis and HIF-1α activity under insulin resistant conditions.

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The family of cytohesins, which are small guanine nucleotide exchange factors (GEFs), consisting of cytohesin-1, cytohesin-2 (ARNO), cytohesin-3 (Grp-1) and cytohesin-4, has been shown to contribute to cellular adhesion and transmigration of leukocytes as well as adhesion and migration of other cell types. They also seem to play an important role in insulin signalling. In earlier studies, we demonstrated that inhibition of cytohesin-2 (ARNO) reduced vascular leakage in vivo. We discovered that ARNO directly affects VEGFR-2 signalling in endothelial cells by controlling internalization and degradation of VEGFR-2. Furthermore, we discovered that ARNO is important for the phenotypic switch of vascular smooth muscle cells (VSMC). We found that an inhibition or inactivation of ARNO affected the morphology, reduced the synthetic activity and migration of VSMC induced by the adipokine resistin. Furthermore, ARNO was shown to be important for resistin dependent MMP-2 production as well as migration through activation of the JNK/AP-1 pathway and p38 MAPK. An ongoing project aims to characterize the function of the cytohesins 1, -2, -3 and -4 in hypoxia induced permeability and angiogenesis in endothelial cells.

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Cardiopulmonary bypass (CPB) is routinely used in cardiac surgery because it allows temporary suppression of cardiac activity while keeping systemic circulation maintained by redirecting the blood from the heart and lungs and instead pump it through an extra-corporal circuit. Several components of the CPB process, such as the shear forces generated by the pumps, hemodilution, exposure of blood to the artificial surfaces of the circuit and the aortic cross clamping may contribute to the induction of a systemic inflammatory response. Moreover, the CPB may influence the pharmacodynamics of administered drugs. The inotropic drug levosimendan is often used as therapeutic approach perioperatively in cardiac surgery patients with CPB to improve hemodynamic stabilization and to reduce the risk of post-surgical low cardiac output syndrome. In cooperation with the Department of Pharmacy, LMU, we recently established a LS-ESI-MS/MS protocol for therapeutic drug monitoring (TDM) of levosimendan and its metabolites OR-1855 and OR-1896, enabling rapid and simultaneous detection of these compounds in serum. Subsequently, in cooperation with the Department of Anaesthesiology at the LMU University Hospital we detected low total serum concentrations as well as unbound fractions of these compounds in cardiac surgery patients with CPB. Moreover, we investigated the effects of levosimendan and its metabolites on inflammatory endothelial activation and found levosimendan and both metabolites to reduce reactive oxygen species, while only levosimendan impaired endothelial adhesion molecule upregulation. An ongoing study investigates if the low concentrations of levosimendan and metabolites, which were measured in cardiac surgery patients, still have protective effects on endothelial activation and vascular permeability. We are furthermore interested in studying the underlying cellular mechanisms for the enhanced endothelial barrier disruption and endothelial activation and dysfunction in patients with CPB.

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Gene delivery to cells constitutes an efficient way to specifically modulate signalling pathways and is a promising approach to induce physiological responses (gene therapy) in both research and medical therapy. Regarding the vasculature, local gene therapy shows great potential in improving ischemic cardiovascular disease and wound healing, but also to inhibit tumor growth and diabetic retinopathy, to mention a few. Upon intravascular application, gene vectors are distributed systemically and apart from reaching the organ of interest, they are also delivered to other sites, causing undesired side-effects. Other hurdles with this technique yet to overcome are poor gene delivery and expression efficiency at the desired site. We have developed an innovative effective gene delivery approach for in vivo use, which combines high efficiency and site-specificity following intravascular application. By using lipid microbubbles coated with superparamagnetic nanoparticles (magnetic microbubbles, MMB) which are associated to lentiviruses, DNA vectors, or AS-ODN we were able to target these to vessels at a specific site in vivo by applying an external magnetic field and subsequent ultrasound mediated disruption of the MMB ("Ultrasound-Targeted Microbubble Destruction"; UTMD) to enable the delivery of their cargo.?This resulted in high transgene expression at the targeted site, whereas the non-specific delivery at other sites was strongly reduced. We further established a technique to efficiently deliver genes to wounds in the dorsal skin of mice to study hypoxic wound healing angiogenesis by using magnetic nanoparticle associated lentiviruses and external magnetic fields. This technique now enables the investigation of cellular mechanisms during vascular regeneration and vascular remodelling in the intact organ.

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